Recovery of nordihydroguaiaretic acid from plant extracts



Patented June 29, 1948 RECOVERY :OF NORDIHYDROGUAIARETIC ACID FROM PLANTHEXTRACTS V,

Ole Gisvold, Minneapolis, Minn., assignor tol'tgents of The University of Minnesota Minneapolis, Minn, a corporation'oflMinnesotav No Drawing.

This invention relates to the preparation ofrelatively pure extracts of plants of the species Larrea divarz'cata, suitable for use in pharmaceuticals andfoods. Plantsof the species Larrea dzmarzcaia are sometimes also known as Larrea. tridentata or COm'lZea tridentata, and are commonlyknown in the United States as creosote bush. The term Larrea divaricata will be used herein.

This application is a continuation-in-part of m copending application Ser. No. 573,311 filed January 17, 1945,. now Patent No. 2,408,924, grantedOctober 8, 1946.

It hasbeen discovered that plants of the species Lizrrea divarz'catu, contain nordihydrogualaretic acid, acompound of phenolic nature, having the empirical formula 018112204 and. the structure:

I CH CH3 HO on,

According to the Geneva nomenclature, nordihydroguaiaretic" acid may be designated beta, gamma-dimethyl, alphadelta-bis (3,4-dihydroxyphenyl) butane The melting point of this materia1is183184 C. i

In addition to nordihydroguaiaretic acid, plant material of the species Lam-ea divarz'cata also appearsto contain other phenolic constituents, rubber-like substances, pigments, cellulose and variousother complexes-which are undesirable contaminants insofar as concerns many uses of the nordihydroguaiaretic acid component, although some of such other constituents are in themselves useful.

It is an object of the invention to provide a convenient -efiicientn method of separating. the constituents of natural plant material. of the species Larrea divarz'cata, and a particular object to provide methods of making extracts .having a high concentration of nordihydroguaiaretic acid.

The present invention involves an improvement on previous methods of extraction and purification and resultsin the isolation of substantially pure nordihydroguaiaretic acidin good yields and with a minimum of processing.

In general th invention comprises the preparation of a crude extract of nordihydroguaiaretic acid (from a suitable source .material such. as the plant Lcrrecdivarzcata, which crude extract is then purified .to yield crystalline nordihydroguaiaretic acid in accordance .with a special procedure outlinedin further.detailhereinafter.

The crude extract may be prepared "by any iii Application January 5, 1946, Serial No. 639,433

7 Claims. (Cl. 260-619) suitable1z rocedure. I prefer, however; to employ one of the processes outlined in my copending applications Ser. No. 478,321 filed March 6, 194-3,- now U. S. Patent No. 2,421,117, granted May 27,

1947, and 'Ser; N0."490,l49, filed June 9, 1943; now PatentNo; 2,382,475. According to the first men'- tioned application the crude extract may be"prepared as f ollowsz' P The plant material consisting of'small'twigs; leaves, stems, flowering tops; some seed; or the whole plant, ifde'sired, is dried either by" air drying .or artificial drying, Foreign material such plished without difliculty, and the powder is fairly dense. The size of'grindin-g is'preferably' 10to 100 mesh; 20-to 80mesh beingsuitable for'm-ost efiicient' extraction. The powder isyellowishgreen in color and has an odor which isnot -unpleasant -when dilute, but is somewhat disagreeable whenconcentrated." The powderedmaterlal maybe stored'in' tightly closed tins fora period of weeks without appreciable deterioration; The whole plant may; if desired, be extracted but for the "most'efiicient and thorough utilization of the raw material "it should be ground to a powder."

Theplant material is-then" extracted preferably by percolation or continuous extractionwith a"solvent"'capable of extracting the resinous con stituents of the plant material. The-suitability of any prospective solvent may be gauged-by testing an extract solution made with the-solvent in question" for the color reactions characteristic of catecholusing a'ferric chloride test solution (known in the pharmacopoeia as ferric chlorideT; S.) in the presence of an aqueous'solution of'sodium" carbonate test solution. Thus'to determine the suitability of a' prospective solvent; one may make a test extraction and then to'the solvent extract 'add'asmall quantity of ferric chloride TS. in accordance with the usual technique for'testing for the color reactions char-f acteristic of catechol. Where the" desired plant fractions are. present,- they are-revealed evenin very dilute strengths. Thus, a small drop of plant extract-maybe diluted to 15 to 20 cc. with methylalcoholand this is used for the color tests. 'It' may be'explained that the characteristic green blue-violet color changes characteristic of catechol may not be very distinct when ferric chloride T. S. alone is used, and so as to bring out the'color reactions (and thus make the test 3 more certain) there is added a dilute, preferably freshly prepared, solution of sodium carbonate. This is added drop by drop to the color test solution after ferric chloride T. S. is added. Where the plant extract solvent in question is suitable for the extraction, the much diluted sample of extract solution will be fairly clear and only slightly tinged, a faint olive upon addition of ferric chloride T. S. Upon addition of sodium carbonate, drop by drop, the color changes to an emerald green (a critical color phase, easily passed). As more sodium carbonate is added, the color changes through to a deep blue-green to blue, blue-violet and finally deep violet-red (wine) color. Where the prospective plant extraction solvent is not suitable, these characteristic reactions do not occur.

The solvent may thus be characterized as one capable of yielding an extract of Larrea divaricata giving the color reactions characteristic of catechol using ferric chloride T. S. aqueous ferric chloride solution) in the presence of sodium carbonate (1% freshly prepared aqueous sodium carbonate solution).

Exemplary suitable solvents are in general organic solvents containing a halogen, oxygen or nitrogen in the molecular structure or mixtures of these solvents. Thus, oxygen containing organic solvents such as the alkyl ethers, notably ethyl ether, isopropyl ether, butyl ether, and the like, cyclic ethers such as dioxane or a ketone such as acetone, may be used. Other oxygen containing organic solvents are the alcohols, such as methyl, ethyl propyl, amyl, isopropyl and isoamyl alcohols or the glycols, such as propylene glycol; alkyl' esters, such as ethyl acetate, or other low boiling acetates; acids such as glacial acetic acid, all are suitable. Likewise the halogen containing organic solvents such as chloroform, dichloromethane, methylene dichloride, ethylene dichloride and ethylene dibromide may be used. Similarly the nitrogen'containing organic solvents such as pyridine and morpholine, nitro-methane, nitro-ethane and nitro-propane may be used. Ethyl ether is preferred where suitable equipment is available.

Some of the foregoing solvents are more efficacious than others and widely different extraction. periods are therefore necessary. Thus, when using di-ethyl ether, the extraction is as complete as necessary for practical purposes in 1 hours to 4 hours, Whereas some of the halogenated solvents, chloroform, ethylene dichloride and the like, require from 8 hours to a day or more for extraction.

The extract is then filtered to remove the extraneous material and the solvent is partially or completely removed by evaporation or distillation. The solvent removal step, the temperatures of distillation or evaporation, and other details depend upon the characteristics of the particular solvent used and are obvious once the solvent has been selected as hereinbefore taught. Thus where ethyl ether is used, simple distillation or evaporation is sufficient for its removal. For economy, the solvent should be recovered.

l After removal of all or nearly all of the solvent there remains a dark, yellowish-brown, tarry mass which is designated herein as the crudef or primary extract, This extract contains substantial amounts of nordihydroguaiaretic acid (probably -35%), and some fats, waxes, rubher-like materials, pigments, some of which are flavone-like materials, and other fractions. It may be pointed out at this juncture that there is no accurate method available for the quantitative determination of nordihydroguaiaretic acid, and the content is therefore judged from the amount of crystalline nordihydroguaiaretic acid recovered, as hereinafter described from a given quantity of the primary extract.

According to the later application referred to above the crude extract may be prepared as follows:

There may be utilized the whole plant, including the entire woody portions, leaves, stems, fiowers and buds. The plant material may be used either in the dried or green states, the lattere being preferred. The plant material is preferably not ground, although chopped or packed plant material may be used for convenience in handling.

The plant material is extracted with an aqueous solution of alkali hydroxide such as sodium hydroxide, potassium hydroxide or with ammonium hydroxide. Sodium and potassium hydroxides are preferred because of their excellent extractive ability, loW cost and availability. The aqueous hydroxide solution may be used either weak or strong, preferably a concentration of from 2 to 10%, and to the aqueous solution there is added a reducing agent, preferably sodium hydrosulfite, the percentage of the latter being about half of the percentage of the hydroxide, viz. 1-'5%, although this is not critical. The reducing agent serves to protect the ortho hydroquinone nuclei of the nordihydroguaiaretic acid from oxidation during the alkali extraction. Sodium hydrosulfite serves admirably for this pur-. pose but other reducing agents of equivalent strength, capable of reducing orthoquinones to the corresponding orthohydroquinones under alkaline conditions, may be used. The extraction is made at room temperature, viz. 15-25 C. al-

though slightly elevated temperatures are not harmful.

Extraction is carried out by leaching or percolation, and is usually complete in from 15 minutes to 1 hour. If desired, the extraction may be carried out by a counter-current procedure in a number of vessels, the aqueous hydroxide-reducing agent solution being removed from the first Vessel and conveyed onto the plant material of the second vessel after a few minutes extraction in'the first vessel. Thereafter, a fresh sodium hy droxide-reducing agent solution is conveyed to the first vessel and after a suitable interval the leaching solution of the second vessel is removed and conveyed into a third vessel containing fresh plant material while the aqueous hydroxide-reducing agent solution is likewise conveyed'from the first vessel to the second vessel. This may be repeated through any number of vessels, the aqueous hydroxide-reducing agent solution being permitted to remain in each vessel for a short time, after which it is removed and conveyed to the next succeeding vessel in the series. After from 2 to 4 leachings of the plant material in the first vessel, the extraction of the plant material therein is complete and it is removed and discarded whereupon fresh plant material is placed in this vessel and it becomes the last in the series. By this counterfiow procedure, efficient use may be made of the aqueous hydroxide-reducing agent solution, 7

The leaching liquid containing the extracted material is then acidulatedwith any suitable inorganic or organic acid, hydrochloric, sulfuric and glacial acetic acids being exemplary. It is desired that the acidulation be carried to the point at which a flqceulatiomaiinears inythe -.ext.raction solutions... ati'whichr point: thezsolutiomis. dust. neutrallor a; trifleebasic when;testedqwithditmusi; Init v nt; toosmnchc cidais; addedra. con iderable: apre itationewillioccunbutthere stillremainsanappreciabletdispersionof colloidalparticles; However,

norharmaisdoneby over acidulatiom and should. suchnbe. en.cuntered,:,it :is;only necessary to, add a dilute .alkali ,solution. to;;decrease the acidity slowly the. fiocculationa again appe rs. A13

theeflocculation stage,;:there ,still remains in the extraction liquor a considerablejiiamountof,sus -r1 pended matter which imparts to .the.-liquor,..a somewhati..cloudy appearance However,; the fiocculent precipitate ,.represents .a good portion ofwthel desiredconstituents and thissettles out af-tenstandingc-fon12.hours or more.- Dueto con-, I siderationnoftcost .of raw material it is usually unnecessary to recover the suspended matter. in theyextractiomliquor but :this .may be. recovered andlused-if desired.

Them-aqueous. extraction solution, neutralized asindicated', may also-be treated by centrifuging Crude. :extracts thus ,prepared- .or prepared .by. other: procedures which resultin crude extracts of the same type, can then .be further processed. inaccordance with .the following. method A tsolution-of .crude nordihydroguaiaretic acid together withwaccompanyingv. impurities is prepared ibye dissolving the crudermaterial. in ..any suitabletorganic solvent; Asthesolvent for the crudeextract-at this stage, Ijprefer to use ethyl ethern-or isopropyl ether, although: other ethers such asbeta, betadichlor-diethyl ether or di chlor-dimethylether serve almost as well, and being less volatile present less fire hazard. These latter'ethers are, however, much-more expensive. Themsolutionlof..crude extractethus obtained is then treated with an alkaline material, such as sodium carbonate, in a finelyidivided solid form, ore-where. the solution of crude extract is immiscible in water it may be treated with an aqueous solution of the alkaline material; Thetreatment with the alkaline materiahmay be conducted :in any suitable manner as by suspendingflandagitating the solid alkaline material in. the solutiont-of crudeextractfor a short period. oftimelfollowed by separating the solid from the solutiom, As an alternative the solution of crude extract. may be passed through a column of the solid;preferablyimixed with an inert agent such as;,fi;lter.;cell.

The-treatment .withan aqueous solution, of the alkaline material-may be effected in any ,usual The alkaline material removes pigment impuritieswpresent in the acrude extract, some of which-at least mightfind their way into the final product;were it. notior. this treatment. The treatmentyishighlyefficaciousyand results in a product; far; superior I to; those; heretofore avail able. and at the same time-, involves a minimum,

ip mes i .Hi h y ldsnf arhieh p r y prodr'w teammate-iced:

t l ati m. .(preferably l unden: vacuum). .toremoy most, ,if not- .all the solvent :and. then; mixedzwit .1 & fluan tyi f a halo enated.hydrocarbon to; for

a homogeneous solution-. The, removal... of ;;.th original solvent may .be accomplished .ingamtt'ofnt a numberof suitable ways, For example, wheres-r the ,original solvent is;,more volatile, than-.;.th p

i0 halogenated hydrocarbon, it may beremovedi,

simple distillation. Where the original ,solven is water miscible. it may beremoved bywashing with water. Other suitable methods of removing the original solvent will beapparent to those skilled in. the art. Thesolution of nordihydro:

guaiaretic acid in halogenated hydrocarbon .re.-. maining aftertheremoval of the original solvent, is then concentrated toa small yolume atswhich point it has the consistency of a thinsyrup, Thea mixture is cooledand stirred, and. crystallization The crystallization of nordihydroguaiae retic a-cid usually. occurs in a few minutes but.may,

The semi-1e.-

solid crystalline mass is composed-of crystallized...

begins.

require 24-48110111'3 to be complete.

nordihydroguaiaretic acid and dissolved impurie.

"ties, principally phenolics, and a small amount oft}. halogenated hydrocarbon. The semi-solid mass...

is then treated with a small quantity of. preferably pure nordihydroguaiaretic acid in a good yield.

the invention? Example I A primary extract from 200 .grams of drug madewith sodiumhydroxide and sodium hydro s sulfite in accordance with my preceding applica-t tion'SerialNo,490,149, now Patent No. 2,382,475p

I was-covered with150 cc. ofisopropyletheru This-"- mixture was stirred vigorously for about 10min Lites. If the. primary extract has not been thorl oughly acidulated in its preparation, 1 to 3 cc. oft strength connnercial hypophosphorus acid r can beaddedto. this mixture for its protectingeffect.upon..nordihydroguaiaretic acid. The supernatant liquid was decanted and the residuetreated with a second 150 cc. portion of isopropyl-m The two portions of ether. as described above. solvent were combined, treated with grams 60.;The ether solution wasthen decanted. The highly colored sodium carbonate was washed with two 25 cc. portions of isopropyl ether. These wash-.-,

ings were combinedwith the original ether solution;and then sti rredyigorously with 2 .or 3 grams 1 of. sodium hydrosulfite (powdered) cc. of ethen,

removedand, about-5 cc. of ethylene dichloride re-- inlaineil The extract was.:then cooled andstirretl;

75. Crystallization -..of nordihydroguaiaretic acid:.toolr l The following specific examples, which are not... be construed as limiting, will serve to illustrate .of finelydivided solid sodium carbonate and the mixturestirred rapidly .for .a few minutes. This steperemoves some resinous material andvthe pigments that have. a tendency subsequently to crystalilze with the nordihydroguaiaretic. acid:

The mixture was filteredand the filtrate concentrated; undenayacuum untilv the mixture wasthe consistency of a thin syrup containing about 5 to. 10,0:

10 to cc. of ethylene dichloride- 0 were: thenadded and. themixture heated on the steam; bathuntilthe extract had a consistency'ofs a thin syrupr. At this pointcthe ether had been once with 25 cc. of isopropyl ether.

place in a few minutes. The mass was allowed to stand several hours to insure complete crystallization of thenordihydroguaiaretic acid. The mass was then gently warmed on the steam bath and to cc. of warm ethylene dichloride were added and the mixture thoroughly stirred. The mixture was then filtered by means of suction and the crystalline material washed with successive portions 5 cc. each of hot ethylene dichloride (total 25-50 cc.). The nordihydroguaiaretic acid that remained on the filter paper was finely crystalline, light in color, and-of excellent quality.

Example II A primary extract from 200 grams of drug, made with sodium hydroxide and sodium hydrosulfite in accordance with my preceding application Serial No. 490,149, now Patent No. 2,382,475, was covered with 150 cc. of isopropyl ether. This mixture was stirred vigorously for about 10 minutes. If the primary extract has not been thoroughly aci-dulated in its preparation, 1 to 3 cc. of.

50% strength commercial hypophosphorus acid can be added to this mixture for its protecting effect upon nordihydroguaiaretic acid. The supernatant liquid was decanted and the residue treated with a second 150 cc. portion of i'sopropyl ether as described above. The combined ether extractions were shaken with two 200 cc. portions or 5% sodium carbonate containing 2 /2-% sodium hydrosulfite to remove certain resinous material and pigments that have a tendency subsequently to crystallize with the nordihydroguaiaretic acid. The sodium carbonate washings were washed This was combined with the above ether solution and the combined solutions washed once with 20 cc; of water and once with 20 cc. of 5% hypophosphorus acid. The two portions of solvent were combined, filtered and concentrated under a vacuum until the mixture was the consistency of a. thin syrup'containing about 5 to 10 cc. of ether. 10 to 75 cc. of ethylene dichloride were then added and the mixture heated on the steam bath until the extract had a consistency ofa thin syrup. ,At this point the ether had been removed and about 5' cc. of ethylene dichloride remained. The extract was then cooled and stirred. Crystallization of nordihydroguaiaretic acid took place in .a few minutes. The mass was allowed to stand several hours to insure complete crystallization of the nordihydroguaiaretic acid. The mass was then gently warmed on the steam bath and 5 to 10 cc. or warm ethylene dichloride were added and the mixture thoroughly stirred. The mixture was then filtered b means of suction and the crystallinelmaterial washed with successive portions 5 cc. each of hot ethylene dichloride (total 25-50 cc). The nordihydroguaiaretic acid that remained on the filter paper was finely crystalline, light in color, and of excellent quality. Yield, 5%.

A peculiar phenomenon appears to be involved in the process of this invention. When it is attempted to crystallize nordihydroguaiaretic acid from a solution of it and ordinarily occurring impurities in an ordinary solvent such as alcohol or. ether, no crystallization occurs, but rather the whole mass appears to solidify to an amorphous somewhat plastic body and no separation of the nordihydroguaiaretic acid can be effected. When, however, the crude extract is transferred from its original solvent to the halogenated hydrocarbon solvent, and the solvent partly evaporated, a supersaturated solution appears to result front which the crystalline nordihydroguaiaretic acid can readily be isolated. Apparently the manner in which the supersaturated solution is prepared and the nature ofthe solvent employed affect the balance of the physical system such that a crystalline precipitate is obtained; Whatever the ex planation of the phenomenon, it does occur and provides a simple and economical method of isolating nordihydroguaiaretic acid from a crude extract and involves a tremendous advance over the processes heretofore available for the isolation of this material The ether employed as a solvent for the crude extract before transference to the halogenated hydrocarbon may be any of a variety of lower aliphatic ethers, preferably not higher than butyl ether. Thus, ethyl ether, n-propyl ether, isopropyl ether or butyl ether may lbe used. Similarly mixed others may be used.

A variety of alkaline materials may be employed for removal of resinous materals and pigments. Thus, sodium or potassium carbonates, aluminates and silicates or mixtures thereof or alkali metal salts of similar degree of alkalinity are suitable.

Apparently any halogenated lower aliphatic hydrocarbon which is miscible with the ether solution of the crude extract may be used as the solvent from which the nordlhydroguaiaretic acid is crysta1lized.- Chloroform, propylene dichloride, methylene dichloride and ethylene dichloride are particularly suited and of these, ethylene dichloride appears to possess characteristics best suited for this purpose.

The filtrate containing halogenated solvent and halogenated solvent washings may be collected, combined and concentrated to yield a second crop of nordihydroguaiaretic acid. This procedure may be repeated until no more crystals are obtained. These crops are of lesser purity, ranging from ecru to pale yellow in color, but are suitable for many purposes where a high degree of purity and white color are not essential,

It will be apparent that numerous variations may be made within the scope of the invention without departing from the spirit thereof. For example, time periods, temperatures, amounts of solvents and the like, may be varied from those specifically set forth in the specific examples. It is to be understood therefore that the invention is to be limited only by the appended claims.

What I claim is:

1. Process of separating nordihydroguaiaretic acid from a crude extract containing nordihydroguaiaretic acid and impurities dissolved in a lower aliphatic ether solvent therefor, which comprises contacting the resultant solution with an alkaline material having an alkalinity within the approximate range of that of the alkali metal carbonates, aluminates and silicates, separating the:

alkaline material from the solution, adding a halogenated lower aliphatic hydrocarbon solvent;

of th alkali metal carbonates, aluminates and silicates, separating the alkaline material from the solution, adding a halogenated lower aliphatic hydrocarbon solvent which is miscible with said ether solution, removing said ether solvent and part of said halogenated hydrocarbon solvent, and crystallizing nordihydroguaiaretic acid from the remaining halogenated hydrocarbon.

3. Process of separating nordihydroguaiaretic acid from a crude extract containing nordihydroguaiaretic acid and impurities dissolved in a lower aliphatic ether solvent therefor, which comprises contacting the resultant solution with an aqueous solution of an alkaline material having an alkalinity within the approximate range of that of the alkali metal carbonates, aluminates and silicates, separating the alkaline material from the solution, adding a halogenated lower aliphatic hydrocarbon solvent which is miscible with said ether solution, removing said ether solvent and part of said halogenated hydrocarbon solvent, and crystallizing nordihydroguaiaretic acid from the remaining halogenated hydrocarbon.

4. Process of separating nordihydroguaiaretic acid from a crude extract containing nordihydroguaiaretic acid and impurities dissolved in a lower aliphatic ether solvent therefor, which comprises contacting the resultant solution with a finely divided solid alkali metal carbonate, separating the alkali metal carbonate from the solution, adding a halogenated lower aliphatic hydrocarbon solvent which is miscible with said ether solution, removing said ether solvent and part of said halogenated hydrocarbon solvent and crystallizing nordihydroguaiaretic acid from the remaining halogenated hydrocarbon.

5. Process of separating nordihydroguaiaretic acid from a crude extract containing nordihydroguaiaretic acid and impurities dissolved in a lower aliphatic ether solvent therefor, which comprises contacting the resultant solution with an aqueous solution of alkali metal carbonate, separating the alkali metal carbonate from the solution, adding a halogenated lower aliphatic hydrocarbon solvent which is miscible with said ether solution, remov- 10 ing said ether solvent and part of said halogenated hydrocarbon solvent and crystallizing nordihydroguaiaretic acid from the remaining halogenated hydrocarbon.

6. Process of separating nordihydroguaiaretic acid from a crude extract containing nordihydroguaiaretic acid and impurities dissolved in a lower aliphatic ether solvent therefor, which comprises contacting the resultant solution with a finely divided alkali metal aluminate, separating the alkali metal aluminate from the solution, adding a halogenated lower aliphatic hydrocarbon solvent which is miscible with said ether solution, removing said ether solvent and part of said halogenated hydrocarbon solvent and crystallizing nordihydroguaiaretic acid from the remaining halogenated hydrocarbon.

'7. Process of separating nordihydroguaiaretic acid from a crude extract containing nordihydroguaiaretic acid impurities'dissolved in a lower aliphatic ether solvent therefor, which comprises contacting the resultant solution with a finely divided alkali metal silicate, separating the alkali metal silicate from the solution, adding a halo-, genated lower aliphatic hydrocarbon solvent which is miscible with said other solution, removing said ether solvent and part of said halogenated hydrocarbon solvent and crystallizing nordihydroguaiaretic acid from the remaining halogenated hydrocarbon.

OLE GISVOLD.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 1,595,604 Hefiner Aug. 10, 1926 1,986,320 Burdick Jan. 1, 1935 2,043,102 Kester June 2, 1936 2,296,952 Ross Sept. 29, 1942 2,332,475 C isvold Aug. 14, 1945, 

